HDM2 plays a central role in regulating and influencing important cell-signalling pathways. HDM2 is known to interact with a range of different proteins that influence cellular apoptosis, proliferation and survival.
Thus, amongst other proteins, HDM2 binds to the tumor suppressor protein p53 and targets this protein for ubiquitination and degradation, prevents translocation of p53 to the nucleus by facilitating translocation to the microsomes. Thereby, HDM2 prevents transactivation of p53 target genes that are implicated in the regulation of cell cycle and apoptosis. The p53 protein is a potent cell cycle inhibitor that prevents propagation of permanently damaged cell clones by the induction of growth arrest or apoptosis, resulting in the protection against development of cancer by guarding cellular integrity.
Both p53 as well as HDM2 can be associated with cancer: about 50% of all human tumors harbor a mutation or deletion in the p53 gene that impairs normal p53 function (Hollstein et al. Science 1991, 253, 49-53). In many cancers with wild-type p53, HDM2 is overexpressed, disabling the normal p53 function (Momand et al. Nucleic Acids Res. 1998, 26, 3453-3459).
The HDM2 gene has a p53-responsive promoter element and elevated levels of p53 that translocate to the nucleus induce expression of HDM2. Induction of HDM2 by p53 forms an autoregulatory feedback loop, ensuring low levels of both HDM2 and p53 in normally proliferating cells (Michael and Oren Semin. Cancer Biol. 2003, 13, 49-58; Vousden and Lu Nature Reviews Cancer 2002, 2, 594-604). However, in many cancers this normal ratio of HDM2 to p53 is changed and misregulated.
Inhibiting the interaction of HDM2 with p53 in cells with wild-type p53 or mutated p53 should lead to an increase of p53 levels in the cytosole, facilitating normal nuclear translocation of normal or mutated p53, cell cycle arrest and/or apoptosis and restoring the tumor suppressor role of p53. The feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of HDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides).
HDM2 also binds to the tumor suppressor pRB, as well as E2F-1 (Yang et al. Clinical Cancer Research 1999, 5, 2242-2250).
E2F-1 is a transcription factor that regulates S phase entry and has been shown to cause apoptosis in some cell types when overexpressed. HDM2 binds to E2F through a conserved binding region at p53, activating E2F-dependent transcription of cyclin A, and suggesting that HDM2 small molecule ligands or antagonists might have also anti-tumor effects in cells independent of their role of restoring p53 function.
HDM2 can associate in vitro and in vivo with the mammalian Numb protein. The association occurs through the N-terminal domain of HDM2, which is the region also involved in p53 binding. The Numb protein is involved in the regulation of cell fate and in a variety of developmental processes, most notably in the nervous system. Through its interaction with Numb, HDM2 may influence processes such as differentiation and survival. This could also contribute to the altered properties of tumor cells, which overexpress HDM2 (Juven-Gershon et al. Mol. Cell. Biol. 1998, 18, 3974-3982).
There is also evidence that HDM2 has a direct role in the regulation of p21, a cyclin-dependent kinase inhibitor. The inhibition of HDM2 with anti-HDM2 antisense oligonucleotide or Short Interference RNA targeting HDM2 significantly elevates p21 protein levels in p53 null PC3 cells. In contrast, overexpression of HDM2 diminishes p21 levels by shortening the p21 half-life, an effect reversed by HDM2 antisense inhibition. HDM2 facilitates p21 degradation independent of ubiquitination and the E3 ligase function of HDM2. Instead, HDM2 promotes p21 degradation by facilitating binding of p21 with the proteasomal C8 subunit. The p21 and HDM2 bind through 180—the 298 amino acids region of the HDM2 protein (Zhang et al. J. Biol. Chem. 2004, 279, 16000-16006).
There is also evidence for a malfunctioning HDM2 regulation having effect on a proper p53 function and causing cancer, beyond mutated p53 or overexpression of HDM2. Thus, when E2F signals the growth of a cancer, P14ARF is dispatched to break down HDM2, freeing p53 to kill the cancer cell. In certain cancers P14ARF is lacking (Moule et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14063-6). P14ARF binds to HDM2 and promotes the rapid degradation of HDM2. ARF-mediated HDM2 degradation is associated with HDM2 modification and concurrent p53 stabilization and accumulation.
The validity of inhibiting HDM2 as a therapeutic concept has been first demonstrated by antisense HDM2 inhibitors that exhibit significant antitumor activity in multiple human cancer models with various p53 statuses (Zhang et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 11636-11641).
Small molecule antagonists of the HDM2 protein interactions may therefore offer a viable approach towards cancer therapy, either as single agents or in combination with a broad variety of other anti-tumor therapies.
There is also growing evidence that HDM2 plays an important role in viral infections. First, it is known that viruses rely on changing normal p53 signalling (O'shea and Fried M. Cell Cycle 2005; Machida et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 23, 101, 4262-7).
Second, HDM2 directly interacts with viral proteins, for example HDM2 is a target of simian virus 40 in cellular transformation and during lytic infection (Henning et al. J. Virol. 1997, 71, 7609-7618). Furthermore, the HDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of HDM2 by SV40 is aimed at preventing HDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells. A trimeric LT-p53-HDM2 complex is formed with simian virus 40 large tumor antigen (LT) in SV40-transformed cells.
The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat. HDM2 has been shown to interact with Tat and mediating its ubiquitination in vitro and in vivo. In addition, HDM2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination (Bres et al. Nat Cell Biol. 2003, 5, 754-61).
Small molecule inhibitors of the HDM2 interaction have been reported and show pro-apoptotic effects in in vitro models and an antitumor effect in animal models of cancer. Thus, benzodiazepines have been used as a chemical scaffold to achieve HDM2 inhibitory activity (Grasberger et al. J. Med. Chem. 2005, 48, 909-912; Parks et al. Bioorganic & Medicinal Chemistry Letters 2005, 15, 765-770). Similarly, imidazolines (Vassilev et al. Science 2004, 303, 844-848), isoindolones (Hardcastle et al. Bioorganic & Medicinal Chemistry Letters 2005, 15, 1515-1520), norbornanes (Zhao et al. Cancer Letters 2002, 183, 69-77) and sulfonamides (Galatin and Abraham J. Med. Chem. 2004, 47, 4163-4165) have been reported as small molecule HDM2 inhibitors.
It has also been reported that HDM2 ligands have a cytoprotective effect. Thus, HDM2 inhibitors can be employed in methods of inducing cytoprotection and are useful to protect non-target cells against the harmful effects of chemotherapeutic agents. The amount of HDM2 inhibitor that provides such an effect can be about 5 to about 10 fold lower than the amount needed to induce apoptosis (Koblish et al. WO03095625, METHOD FOR CYTOPROTECTION THROUGH HDM2 AND HDM2 INHIBITION, 2003-11-20).
Isoquinolones have been reported already as potent antagonists of the platelet glycoprotein IIb-IIIa (Fisher et al. J. Med. Chem. 1997, 40, 2085-2101) to treat cardiovascular diseases.
Pancrastatin is a naturally occurring alkaloid with an isoquinolone structure exhibiting anticancer properties, by acting on the tubulin cytosceleton. Lysolipin and Cervinomycin are antibiotics isolated from streptomyces violaceoniger. Lycoricidine and narciclasine are isoquinolone based plant-growth regulators, Gliquidone is an antidiabetic medication which is used in those patients with adult maturity onset or non-insulin dependent diabetes (NIDDM). It works by lowering blood sugar levels by stimulating the production and release of insulin from the pancreas. It also promotes the movement of sugar from the blood into the cells in the body which need it. Tesicam is an isoquinolon-dione used for its anti-inflammatory properties. These compounds have low toxicity, good pharmaco-kinetic properties and render the chemical class of isoquinolones an interesting scaffold for new drug candidates.
In this present invention, we describe novel, isoquinolone scaffold based small molecules that are inhibitors of HDM2 and can be used as novel therapeutic agents.